Reproduction (2001) 121, 207–216 Review

Significance of incorporating measures of sperm production and

function into rat toxicology studies
Sally D. Perreault1 and Aida M. Cancel2
1Reproductive Toxicology Division, NHEERL, US EPA, Research Triangle Park, NC 27711,

USA; and 2Toxicology Program, University of North Carolina, Chapel Hill, NC 27599, USA

The rat is the preferred species for reproductive toxicity testing. The inclusion of
measures of rat sperm quality, such as motility and morphology, into reproductive test
protocols often increases the sensitivity of the test to detect effects, and provides the
toxicologist and risk assessor with valuable information about the nature of the repro-
ductive toxicity of the test substance. Technical advances in computer-aided sperm
analysis have made it possible to evaluate motion characteristics of rat spermatozoa.
This technology can provide an objective means of classifying the motion of rat sperma-
tozoa as progressive or non-progressive, as required in test protocols. More specific tests
of rat sperm function are being applied for the purpose of evaluating modes and mecha-
nisms of toxicant action. Computer-aided sperm analysis can be used to evaluate sperm
motion during cultures that support sperm capacitation and to identify hyperactivated
spermatozoa. Under the same culture conditions, acrosome-specific stains can be used
to identify effects of toxicants on the acrosome reaction. These approaches, in combina-
tion with in vitro fertilization in rats, can pinpoint sperm functional deficits and thereby
assist the toxicologist in addressing hypotheses regarding the cellular–molecular bases of
toxicant-induced male infertility.

Male reproductive toxicology is a relatively new field that
has emerged in part as a consequence of reports in the late
1970s that male workers became infertile after being
exposed to the pesticide dibromochloropropane (DBCP)
(reviewed in Lahdetie, 1995). Epidemiological investiga-
tions involving the examination of semen samples pro-
vided by these men revealed that their infertility was due
to azoospermia or diminished sperm counts. Thus, semen
analysis, which had been an investigational tool for the
infertility physician for many years, became a means for
investigating the potential for drugs and chemicals to affect
male reproductive health.
The widely publicized DBCP story served to alert regu-
latory agencies, as well as the public, about the potential
for toxicants in the workplace or the environment to alter
reproductive function in men. It also provided the ratio-
nale for adding direct evaluations of sperm production and
quality to reproductive toxicology test protocols in which
the rat is the preferred test species. Here we review the sig-
nificance of including data on rat sperm production and
function, such as epididymal sperm reserves, motility and
morphology, in toxicology testing. In addition, we discuss
the development and application of more specific tests of
rat sperm function as may be needed to elucidate the
Email: darney.sally@epa.gov
modes and mechanisms of toxicant action relevant to esti-
mating reproductive risks in humans.
Sperm measures as endpoints in reproductive test
protocols
It is important to understand the context in which sperm
measures are used as markers of male reproductive effects
in toxicology testing. Federal agencies, such as the
Environmental Protection Agency (EPA) and the Food and
Drug Administration (FDA), and international bodies, such
as the Organization for Economic Cooperation and
Development (OECD), provide a number of test protocols
and guidelines for identifying adverse reproductive effects
(reviewed in Blazak, 1997; Clegg et al., 2001). These pro-
tocols and guidelines are used by industry to test pesti-
cides, industrial chemicals, pharmaceuticals and food
additives for potential reproductive toxicity in laboratory
animals. The rat is the animal of choice for these tests, in
part because there is a large toxicology database for this
species. If a substance is identified as a reproductive toxi-
cant in rats, further studies are conducted to determine the
level at which it is toxic, the likelihood that it will be toxic
to humans as well as to rats, and the extent to which
humans might be exposed to the substance. All of this
information is critical for performing a risk assessment on

© 2001 Journals of Reproduction and Fertility

1470-1626/2001
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208 S. D. Perreault and A. M. Cancel
the chemical and, ultimately, for making regulatory deci-
sions about the allowable uses of the substance as well as
labelling requirements.
The most comprehensive test for reproductive toxicity is
the multigenerational test, which is designed to detect
adverse effects of exposure at all stages of the reproductive
life cycle, as well as potential transgenerational effects (re-
viewed in Blazak, 1997; Clegg et al., 2001). Young adult
rats of both sexes (the parental or F0 generation) are ex-
posed to the test chemical daily for 10 weeks, a period suf-
ficiently long to ensure that effects on germ cells exposed
at any stage of spermatogenesis will be detected when that
animal is mated. The animals are then mated with each
other and pregnant dams are dosed through pregnancy,
birth of the first (F1) generation and lactation. After wean-
ing, selected F1 pups are dosed into adulthood and
throughout breeding, pregnancy and lactation to give rise
to a second (F2) generation that is exposed until weaning.
The multigenerational test relies heavily on measures of
reproductive performance such as breeding, fertility and
litter size, with additional information provided by
reproductive organ weights and routine histology mea-
sured in each generation. Since both partners in breeding
pairs have been treated, it is frequently impossible to as-
certain the affected sex and, since treatment is prolonged,
little information is provided regarding specific cellular tar-
gets. A variation of this test, the reproductive assessment
by continuous breeding (RACB) test used by the National
Toxicology Program to evaluate high priority chemicals,
includes serial breeding during the treatment phase in the
parental generation. The RACB test obtains additional in-
formation about the onset and severity of infertility and
includes the option of crossbreeding with untreated ani-
mals to determine the affected sex (Chapin and Sloane,
1997; Clegg et al., 2001).
In 1998, as part of an interagency harmonization effort
and after much deliberation, the standard multigenera-
tional test protocol was revised and updated to include
direct measures of sperm production, motility and mor-
phology (US Environmental Protection Agency, 1998;
Chapin and Conner, 1999; Claudio et al., 1999). These
measures had been included in the RACB test, initially
using mice but more recently using rats (Chapin and
Sloane, 1997; Chapin et al., 1997) and proved useful in
numerous other reproductive toxicology studies in rats, es-
pecially those involving shorter-term exposures (Harris et
al., 1992; Linder et al., 1992; Clegg et al., 2001). This arti-
cle focuses on the significance of sperm measures, but the
harmonized guidelines also emphasize the importance of
optimal fixation of the testis so that subtle changes in testis
histopathology can be detected (Russell et al., 1990;
Chapin and Conner, 1999). The guidelines also include
morphological measures of sexual differentiation and
puberty that are sensitive indicators for environmental
oestrogens and other hormone mimics (Claudio et al.,
1999; Clegg et al., 2001). As currently structured, the har-
monized multigenerational test and the RACB test are rec-
ognized as highly effective, apical tests for hazard identifi-
cation when screening pesticides, drugs and industrial
chemicals for effects on reproduction and fertility.
Spermatid counts in testes and epididymides
Information about sperm production and storage is ob-
tained when rats are killed at the end of each generation
in the multigenerational tests, or at specified time points
in short-term protocols. In general, one testis and epi-
didymidis is fixed for histologic evaluation, while the other
is used for determining sperm measures. Sperm production
by the rat testis is conveniently measured by counting the
number of condensed spermatids in a testis homogenate,
expressed as the number per testis or per gram of testis
(Robb et al., 1978). In turn, the number of spermatozoa
per testis can be divided by 6.1 days (the period that sper-
matids reside in the testis after their chromatin condenses
and they become resistant to disruption by homogeniza-
tion or sonication) to derive the sperm production rate
(number of spermatozoa produced per day). Likewise,
mature spermatozoa are collected from the cauda
epididymidis and counted to determine cauda epididymal
sperm reserves. Computer-assisted counting of sperm nu-
clei stained with a DNA-specific fluorochrome (Strader et
al., 1996) can enhance the efficiency with which these
measures are obtained.
Baseline data accumulated in control (untreated) rats in-
dicate that these measures are dependent on age in young,
pubertal rats, but are stable in adult rats (Robb et al., 1978;
Working and Hurtt, 1987; Blazak, 1997) and so make good
indicators of impaired testis function or altered epididymal
capacity. After reviewing the RACB data for 72 studies in
mice, Chapin et al. (1997) concluded that epididymal
sperm count was related linearly to fertility. When com-
bined with data on either sperm motility or sperm morphol-
ogy (both of which exhibit fertility thresholds), much of the
variation in fertility could be explained (r = 0.77). Whether
these relationships hold in rats will be determined as more
data for these endpoints become available from multigener-
ational studies using the new protocol.
Numerous examples from both long- and short-term
studies demonstrate that significant alterations in testicular
and epididymal spermatid counts can occur at doses of
toxicants that are lower than those associated with sterility
(Linder et al., 1992; Clegg et al., 2001). Thus, when
interpreted with other data from fertility and histopatholog-
ical examinations, sperm counts are useful for corroborat-
ing anti-fertility effects, elucidating the mode of toxicant
action, detecting effects at low doses and helping to
determine the affected sex.
Evaluation of sperm motility in rats and the use of
computer-assisted sperm analysis (CASA)
Sperm motility is a requirement for fertility. Therefore, it
is often useful to examine epididymal spermatozoa to
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 Measures of rat sperm function in toxicology studies
determine whether changes in sperm motility account for
changes in fertility status. The harmonized test protocols
specify that epididymal spermatozoa will be examined to
determine the ‘percentage of progressively motile sperma-
tozoa.’ The intent of the test is to determine whether sperm
samples recovered from treated animals differ significantly,
with respect to their motion, from those recovered from
untreated control animals.
The use of computer-assisted sperm analysis (CASA)
makes it practical to evaluate the motion of a large num-
ber of spermatozoa in a short time. Microscopic images of
spermatozoa are detected by video technology and a com-
puter ‘captures’ each video image, records the location of
the sperm head in each video frame and reconstructs the
sperm path by connecting those images. Typically, today’s
instruments capture the maximum number of images pos-
sible according to the video standard in use (60 images s–1
in the US and 50 images s–1 in Europe), and then generate
values for seven or eight characteristics for each track (for
a technical review, see Boyers et al., 1989), each of which
provides information about one aspect of sperm motion,
such as vigour, velocity or swimming pattern.
CASA technology was first applied to rat epididymal
spermatozoa by Working and Hurtt (1987) and its poten-
tial advantages in terms of efficiency and objectivity were
immediately attractive to the toxicology community,
which began to explore the usefulness of this technology.
It soon became clear that methods for sample collection,
preparation and handling (for example, culture medium
and temperature), as well as chamber depth and instru-
ment settings, can give rise to a great deal of intra- and
inter-laboratory variation in values obtained by CASA
(Slott et al., 1991; Chapin et al., 1992). Workers using
CASA convened to discuss these issues and published
guidance on the conduct of CASA for rat spermatozoa
(Seed et al., 1996). Improvements in the optical systems
and capture algorithms in CASA systems have increased
the accuracy of rat sperm tracking, and more powerful
computers have made it possible to track spermatozoa for
longer periods, even continuously (Moore and Akhondi,
1995). These advances have improved the consistency of
the data, and CASA is now used routinely to monitor rat
sperm motion in many toxicology laboratories (Perreault,
1998).
Although Federal test guidelines do not require the use
of CASA, the method is well suited for defining ‘progres-
sively motile spermatozoa’ on the basis of selected CASA
parameters. Freshly diluted cauda epididymal rat sperma-
tozoa typically swim ‘fast and straight’. Workers using
CASA can set thresholds for two CASA measures, one for
‘fast’ and one for ‘straight’ and require that a spermato-
zoon exceed both to be considered progressively motile.
In this regard, the measure called velocity of the average
path or VAP, and the measure called straightness (derived
from the ratio of straight-line velocity to VAP) can be com-
bined in a bivariate analysis to identify and count progres-
sively motile spermatozoa automatically. For example, we
209
have found that a VAP threshold of 100 µm s–1 and a
straightness threshold of 50 defines about 80% of cauda
epididymidal spermatozoa from untreated (control) rats as
‘progressively motile’ under our sample preparation and
analysis conditions (S. Perreault, S. Jeffay and L. Strader,
unpublished). The percentage of progressively motile sper-
matozoa was found to be the most valuable measure
(when compared with individual CASA parameters) for de-
tecting subtle changes in sperm motion induced by three
model toxicants with different mechanisms of action
(Horimoto et al., 2000).
Information about the percentage of progressively motile
spermatozoa is useful when a chemical affects the percent-
age of motile spermatozoa or the quality of sperm motion.
In many cases, a testicular toxicant, applied for a long pe-
riod, will produce testicular atrophy at the higher dosage,
effectively shutting off the production of spermatozoa, with
consequent infertility. However, at lower dosages, or if the
chemical is being studied after short exposure times to
track the development of the infertility, the adverse effect
may show up as a decrease in the percentage of motile
spermatozoa, the quality of the motion (fewer progressive
spermatozoa), or both. This effect may occur in the ab-
sence of an effect on fertility. For example, several Sertoli
cell toxicants produce testicular atrophy at high doses, but
poor sperm quality, secondary to germ cell sloughing, at
low doses (Richburg et al., 1997; Li and Heindel, 1998). In
such cases, information on sperm numbers, motility and
morphology helps to determine the no adverse effect (and
low adverse effect) doses. Such effects might be missed if
fertility were the only outcome considered.
These measures are also valuable indicators of toxicity
in short-term studies that have been proposed as more effi-
cient and less expensive screens (Harris et al., 1992;
Linder et al., 1992). After only a few days or weeks of ex-
posure, the consequences of the toxicity may not be fully
manifest, but the hazard may be identifiable by changes in
spermatozoa that have reached the epididymis (Harris et
al., 1992). Likewise, sperm motility is a valuable indicator
of toxicity that involves only the epididymis or the mature
spermatozoa during epididymal transit (Perreault, 1997;
Klinefelter and Hess, 1998; Perreault, 1998).
Although test guidelines call for only one measure of
the quality of sperm motion (that is, the percentage of pro-
gressively motile spermatozoa), specific CASA outcomes,
such as straightline or curvilinear (point-to-point) velocity
or track amplitude, may provide additional information,
individually or in multivariable analyses, on the pattern of
motility and how it might be altered specifically by a given
toxicant. These specific CASA outcomes are also useful for
monitoring changes in sperm motion that occur during
epididymal maturation in vivo and in vitro (Yeung et al.,
1992; Klinefelter, 1997). In these cases, the mean (or me-
dian) is calculated for a population of spermatozoa (usu-
ally 100–200) from each animal. Consideration of the
distribution of each CASA endpoint within animals may
also be important for detecting specific exposure-related
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210 S. D. Perreault and A. M. Cancel
changes (Toth et al., 1989), as well as changes in motion
associated with the fertilizing spermatozoon in the female
tract. However, it is important to consider that several of
the routine CASA outcomes are highly correlated with
each other and are not truly independent measures. A
novel means for calculating three CASA outcomes, one
that estimates straight line velocity, one that estimates lin-
earity, and a third that indicates predictability of the path,
has been introduced and demonstrated to comparably
(and perhaps more reliably) describe rat sperm motion
(Dunson et al., 1999). Now that CASA technology has ma-
tured, more meaningful ways of handling the large amount
of CASA data are emerging. Valid statistical evaluation will
be critical for demonstrating the ultimate utility of sperm
motion data in risk assessment.
Evaluation of sperm morphology in rats
Typically, rat sperm morphology is evaluated by examin-
ing either wet preparations of fixed epididymal spermato-
zoa using phase-contrast microscopy, or air-dried, stained
smears using conventional light microscopy (Linder et al.,
1992; Seed et al., 1996; Chapin and Conner, 1999).
Untreated rats usually exhibit very few morphological ab-
normalities (< 2%) and examination of 200–500 spermato-
zoa per rat is recommended to detect changes in this
outcome. Each spermatozoon is scored as normal or ab-
normal, and atypical forms are classified by abnormality in
a manner that distinguishes head, midpiece and tail de-
fects (Linder et al., 1992). Head abnormalities described in
the literature include: blunt hook, banana-head, amor-
phous head, pinhead, and double head (Seed et al., 1996).
However, it can be difficult to distinguish borderline forms
with confidence. Automation of sperm morphology mea-
surement using CASA is more difficult for rat spermatozoa,
with their hook-shaped heads, than it is for round or oval
headed spermatozoa. However, one system has been de-
scribed (Davis et al., 1994), and commercially available
programs are currently being developed and validated.
As with motion analysis, sample preparation is critical.
The spermatozoon must lie very flat to ensure accurate as-
sessment of the hook. Any other orientation can introduce
visual artifacts and result in misclassification of normal
spermatozoa as abnormal. Subtle differences such as
straightened hooks can be indicative of immaturity or pre-
mature spermiation, while blunted hooks can be indicative
of abnormal acrosomes or acrosomal degeneration. More
obvious forms, such as amorphous head shapes and dou-
ble or fused spermatozoa, are easier to identify and may
represent more biologically significant malformations
(Linder et al., 1997). As with sperm motility measures,
sperm morphology data can provide valuable insights re-
garding the dose response to a chemical. In addition, mor-
phological deficits, such at detached heads and abnormal
tails, may correlate with sperm viability and be indicative
of cell death after spermiation.
Interpretation of sperm measures in rats with
respect to similar measures obtained in exposed
humans
Several differences in methods and physiology need to be
considered when extrapolating effects in rats to humans, or
comparing the results of rat toxicology and human epidemi-
ology studies. Rat spermatozoa are typically collected from
the cauda epididymidis (or proximal vas deferens) whereas
human spermatozoa are typically evaluated in seminal fluid
(after ejaculation). Dilution of rat spermatozoa into culture
medium could dilute any toxicant remaining in the epididy-
mal fluid, and stimulation of sperm metabolism by energy
substrates in the medium may overcome adverse effects of
toxicants in epididymal fluid. These factors should be consid-
ered when making direct comparisons between sperm mea-
sures made in diluted rat spermatozoa and in human semen.
Rat spermatozoa are very homogeneous in comparison
with human spermatozoa. In a typical sample from a con-
trol rat, most of the spermatozoa will be progressively
motile, and nearly all will be morphologically normal. In
contrast, human semen samples, even those from fertile
men, typically exhibit a wide range of motility and a low
but variable proportion of morphologically normal forms.
Thus, a small change in a sperm parameter in a group of rats
may be statistically significant as a result of the consistency
of the measures in control rats, whereas a change of similar
magnitude in a group of humans may be indistinguishable
against the high background of abnormal spermatozoa. This
is not to say that rats are more sensitive to the compound,
but rather that detection of change may be easier in rats.
Sensitivity is determined by many other factors.
Since many men with borderline semen quality are
thought to be on the threshold of infertility, a small change
in sperm production or function could shift them into the
infertile status. However, it takes large shifts in sperm count
or quality to render a rat infertile. Nevertheless, information
about sperm quality in rats may allow the detection of ad-
verse effects that occur at low dose, or after short-term ex-
posure, especially in cases in which fertility is not affected.
For many years, clinicians have searched for the best
endpoint in semen that could be used to predict fertility in
individual men. Similarly, when CASA technology came
into use, andrologists argued for a single best CASA para-
meter for predicting infertility. A study undertaken to ad-
dress this question indicated that some sperm measures
may be better correlated with fertility than others, espe-
cially under certain circumstances, but that information is
lost by relying on a single outcome (Zinaman et al., 2000).
Likewise, it is naive to think that a single sperm measure in
rats will be sufficient to detect reproductive toxicity from
any chemical exposure. Different reproductive toxicants
act by different mechanisms to produce an array of effects.
Therefore, it is advantageous to consider all the evidence
from sperm numbers, motility and morphology, as well as
from reproductive organ histology, reproductive behaviour
and fertility, to characterize the toxicity and estimate risk.
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 Measures of rat sperm function in toxicology studies 211

Fusion and
decondensation
ONE WAY

Acrosome
Capacitation reaction
STOP

MALE
FE

Cauda
spermatozoa

Ejaculated
spermatozoa

Hyperactivation

Fig. 1. Post-epididymal events required for fertility. At ejaculation, spermatozoa mix with the acces-
sory sex gland fluid. During their journey through the female reproductive tract, sperm maturation
changes and sperm selection occur. Spermatozoa must travel through the female tract where they at-
tach to the oviduct epithelium and undergo capacitation. During the capacitation process, spermato-
zoa undergo changes in their tail and head membranes. Concomitant with capacitation, spermatozoa
undergo changes in their motility and exhibit hyperactivation. Hyperactivation is thought to assist
spermatozoa in detaching from the oviduct epithelium and penetrating the zona pellucida. Once sper-
matozoa bind to the zona pellucida of the egg (or, in some species, before binding to the zona pellu-
cida), spermatozoa undergo the acrosome reaction. After the acrosome reaction, spermatozoa
penetrate the zona pellucida and fuse with the plasma membrane and decondense in preparation for
fusion with the oocyte genome.

However, evaluating multiple endpoints can lead to a
dilemma. How does one interpret subtle changes in a sin-
gle outcome, especially if it is only a single CASA parame-
ter? The toxicologist looks for a change in the most
sensitive end point and argues that this is the ‘low effect
level.’ The regulator looks at this information and asks, ‘Is
this small change biologically significant? Is it adverse?’
Such questions can be difficult to answer and may require
judgement regarding the redundancy of outcomes. Just as
it does not make sense to rely on a single outcome to pre-
dict fertility, it is important to look at all the outcomes to
determine whether the effects of treatment are real and
make sense in the context of all the information.
Although the clinician tries to predict fertility in an indi-
vidual patient, the epidemiologist looks for convincing dif-
ferences between groups of people. In epidemiology, it is
not necessary to show that a decrease in sperm concentra-
tion or sperm motility in an exposed individual will result
in infertility, but whether groups of men exposed to the
same substance show significant and similar changes in
semen quality. The same is true in toxicology when inter-
preting data from rats.
Development of functional tests for evaluation of
modes and mechanisms of toxicity
At ejaculation, spermatozoa are released from the cauda
epididymidis and mix with the accessory gland fluids to
form semen for deposition into the female tract.
Spermatozoa undergo further maturation changes during
the journey through the female reproductive tract. These
changes are termed capacitation and render spermatozoa
capable of fertilization (Fig. 1). As spermatozoa undergo
capacitation, they exhibit a specialized type of motion
called hyperactivated motility (reviewed in Yanagimachi,
1994). Capacitation is also important in preparing the
spermatozoon to bind to the zona pellucida and undergo
the acrosome reaction, a prerequisite for fusing with the
oocyte membrane and thereby completing the fertilization
process. During these final stages of sperm maturation,
spermatozoa may be susceptible to toxicants in the female
tract. Importantly, these terminally differentiated spermato-
zoa cannot repair any damage (physiological or genetic)
that may arise at this time.
Sperm measures described earlier provide valuable in-

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212 S. D. Perreault and A. M. Cancel

oviduct epithelium (Suarez et al., 1991) and to provide in-

Progressive Intermediate Hyperactivated
VAP 159.0 µm s–1 179.8 µm s–1 219.4 µm s–1
VCL 370.5 µm s–1 496.0 µm s–1 652.9 µm s–1
VSL 137.0 µm s–1 88.0 µm s–1 58.4 µm s–1
STR 86% 49% 27%
LIN 37% 18% 9%
ALH 25.4 µm 23.5 µm 34.6 µm
BCF 21.5 Hz 18.5 Hz 30.0 Hz
Fig. 2. Representative tracks and individual computer-assisted
sperm analysis (CASA) parameters of rat spermatozoa during
culture in vitro under capacitating conditions. Initially, spermato-
zoa swim in a progressive manner but, as they capacitate, their
motion becomes more vigorous and less progressive (intermedi-
ate). Finally, hyperactivated spermatozoa are characterized by
whiplash motion with high vigour and low progression.
Individual sperm tracks were reconstructed from the x,y coordi-
nates of the sperm head at each video frame captured during
CASA analysis. VAP, velocity of the average path (mathematically
smoothed); VCL, curvilinear or point-to-point velocity along the
sperm track; VSL, straight line velocity (total distance travelled
divided by time); STR, straightness, or VSL/VAP ⫻ 100; LIN,
linearity, or VSL/VCL ⫻ 100; ALH, maximum amplitude of lateral
head displacement; BCF, beat cross frequency or the number of
times the spermatozoon crosses its average path. Adapted from
Cancel et al., 2000.
formation about testicular and epididymal function, but
they do not measure sperm functional competence or fer-
tilizing ability. However, many in vitro fertilization tests
and other assays for specific aspects of sperm function
have been developed for clinical use and can be applied
in toxicology (reviewed by Muller, 2000). Various strate-
gies have been proposed for using such tests to evaluate
rat sperm function after exposures either in vivo or in vitro
(Perreault, 1989). However, their application in reproduc-
tive toxicology has been limited to date, due in part to the
lack of repeatable, reliable assays in vitro in rats.
Sperm capacitation and hyperactivation assay
Sperm hyperactivation (HA) has been correlated with
sperm fertilizing ability (Johnston et al., 1994). Hyper-
activated spermatozoa exhibit a whipping motion with
high vigour but low progression. This whipping
motion is believed to help spermatozoa detach from the
creased thrust for penetration of the cumulus and oocyte
zona pellucida (Suarez and Dai, 1992; Stauss et al., 1995).
Therefore, objective evaluation of sperm hyperactivation
may be a prognostic end point to test sperm fertility
(Mortimer, 1997).
The ability of rat spermatozoa to hyperactivate in the
oviduct was first described by Shalgi and Phillips (1988).
Few investigators have studied the movement of rat sper-
matozoa during culture in vitro (Jeulin and Soufir, 1992;
Moore and Akhondi, 1995). Hyperactivated motility of rat
spermatozoa under in vitro culture conditions was con-
firmed by Cancel et al. (2000).
Contemporary CASA methods allow spermatozoa with
high velocity to be tracked accurately and classified as hy-
peractivated on the basis of measures of high vigour (for
example, curvilinear velocity) and low progression (for ex-
ample, low linearity, high amplitude) (Fig. 2). The ability
to monitor a functional endpoint such as hyperactivation
will make it possible to optimize culture media and condi-
tions for in vitro capacitation and fertilization in rats, and
will make it possible to evaluate the extent to which a spe-
cific toxicant or its metabolites may impair the ability of
rat spermatozoa to undergo capacitation and thereby
acquire fertilizing ability.
Sperm capacitation and acrosome reaction assays
The acrosome is an organelle that covers the rostral end of
the sperm nucleus. During capacitation, the acrosome be-
comes capable of undergoing a lytic event called the acro-
some reaction. Functionally, the acrosome reaction is
important because only acrosome-reacted spermatozoa
are able to penetrate the zona pellucida and fuse with the
oocyte membrane (reviewed by Yanagimachi, 1994). The
acrosome reaction may occur spontaneously in culture or
may be induced by a specific protein in the zona pellu-
cida. As with hyperactivation, spontaneous acrosome re-
action can be monitored as an indicator that capacitation
has occurred (Muller, 2000).
Limited information is available regarding in vitro ca-
pacitation and acrosome reaction of rat spermatozoa. Early
studies demonstrated that rat spermatozoa can undergo in
vitro capacitation as evidenced by their ability to fertilize
rat oocytes in vitro (Toyoda and Chang, 1974), and can
undergo spontaneous acrosome reaction during culture as
evidenced by their ability to fuse with zona-free eggs in
vitro (Hanada and Chang, 1976). As with other species of
spermatozoa, a variant of the standard Krebb’s Ringer bi-
carbonate-buffered medium supplemented with bovine
serum albumin supports capacitation of rat spermatozoa in
vitro (Toyoda and Chang, 1974). Subsequent modifications
of the rat IVF medium include use of Hepes buffer and in-
creased calcium content. Woods and Garside (1996) in-
cluded caffeine, hypotaurine and heparin in the medium,
and these three additives have been used individually to
successfully stimulate sperm capacitation or the acrosome

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 Measures of rat sperm function in toxicology studies
reaction in other species. However, the addition of these
three agents concurrently in the capacitation media did
not significantly improve the fertilization capacity of sper-
matozoa. A review of the literature on IVF in rats reveals a
great deal of variability with respect to rates of fertilization
and indicates the need for a systematic effort to optimize
IVF conditions for this species.
Spontaneous acrosome reaction has been monitored
during sperm culture in vitro in many species (Cross and
Meizel, 1989). In some, like hamsters and guinea-pigs, the
acrosome is large and the acrosome reaction can be iden-
tified and monitored in motile spermatozoa using phase-
contrast microscopy. However, in species with small
acrosomes, such as humans and rats, it is necessary to fix
and stain or label the acrosomes to see them. In rats, the
acrosome fits tightly over most of the sperm nucleus and
can be visualized directly only using electron microscopy
(Fig. 3). Although this approach can be used to assess the
acrosomal status of rat spermatozoa (Shalgi et al., 1989),
electron microscopy is technically difficult, labour inten-
sive and expensive. Furthermore, spermatozoa must be at
the right orientation for the acrosome reaction to be de-
tected. Since all spermatozoa cannot be scored, the
method is not well suited for quantifying acrosome reac-
tions in a sperm population.
An alternative approach is to use fluorescent probes to
label the acrosomal components. The antibiotic chlortetracy-
cline (CTC) can be used to monitor the acrosome reaction
and, although the exact mechanism is not known, it seems
to interact with divalent cations (Ca2+) attached to the
membrane surface, with a related change in fluorescence.
Oberländer et al. (1996) used the CTC staining pattern to
evaluate the acrosome reaction during culture of rat sperma-
tozoa in vitro, and described three patterns of CTC staining:
(i) the entire head is labelled (which is thought to be indica-
tive of uncapacitated spermatozoa); (ii) staining is over the
acrosome only (the intermediate pattern is thought to be in-
dicative of capacitated spermatozoa); and (iii) the sperm
head is unstained except at its tip (indicating that it is acro-
some reacted). In addition to being relatively easy to per-
form, this assay has the advantage of providing information
on the dynamics of capacitation and the acrosome reaction.
A potential problem is that the acrosome reaction is a contin-
uous process so readings must be taken repeatedly over time
in culture. In addition, care must be taken to score the cells
quickly since the stain bleaches rapidly after exposure to the
light. Furthermore, it is difficult to assign intermediate pat-
terns to a specific category in an objective manner; in human
spermatozoa up to eight patterns have been described.
Fluorescently tagged lectins have also been used success-
fully to label the acrosomes of many species of spermato-
zoa. The lectin peanut agglutinin (PNA) labels the rat sperm
acrosome in developing spermatids (Martínez-Menárguez et
al., 1982). This lectin is being used successfully to monitor
the rat acrosome reaction under in vitro culture conditions
(Fig. 4; A. M. Cancel, S. Jeffay and S. D Perreault, unpub-
lished). Similarly, antibodies raised against acrosomal
213
Fig. 3. Electron micrograph of a rat sperm head showing the
characteristic hooked shape. The acrosome of the rat spermato-
zoon covers most of the sperm head (Yanagimachi, 1994). Scale
bar represents 1.6 µm.
proteins can be used to label the rat sperm acrosome and
monitor the acrosome reaction using immunofluorescence
microscopy. Examples include anti-DE (antibodies raised
against rat androgen-dependent secretory epididymal pro-
tein DE; Cuasnicú et al., 1984) and anti-IP3R (polyclonal an-
tibody directed against inositol 1,4,5-trisphosphate receptor;
Walensky and Snyder, 1995) antibodies.
Finally, acrosomal enzymatic activity has been used as
an indicator of loss of the acrosome (Salzberger et al.,
1992). This method measures the release of acrosomal
acid phosphatase from rat spermatozoa during prolonged
incubation in rat fertilization media but does not distin-
guish between moribund spermatozoa that release the
enzyme owing to cell death (known as the ‘false acrosome
reaction’) and those undergoing a physiological or ‘true’
acrosome reaction.
Methods for labelling spermatozoa for both acrosome
integrity and sperm viability would help overcome this
problem. Such dual labelling should allow the simultane-
ous evaluation of sperm viability and acrosome integrity in
individual spermatozoa, and thus identify dead spermato-
zoa that have undergone a ‘false’ acrosome reaction.
Any of these methods can now be applied to evaluate
the potential effect of toxicants on rat sperm acrosome in-
tegrity and the ability to undergo an acrosome reaction in
vitro. Interest in this potential mechanism of toxicant
action has been raised by the report that cyclodiene insec-
ticides inhibit the human sperm acrosome reaction in vitro
(Turner et al., 1997).
Only a few chemicals have been shown to target ma-
ture rat spermatozoa directly and thereby impair fertililiz-
ing ability (reviewed by Perreault, 1997). For example, the
antifertility effects of ornidazole are thought to be due to
inhibition of sperm metabolism by an active metabolite;
spermatozoa from treated rats exhibit decreased motility
and reduced ability to penetrate a viscous medium in vitro
(Yeung et al., 1995). These observations indicate that
capacitation may be inhibited. When the acrosome
reaction was monitored during culture in vitro using the
CTC assay, no significant differences in the percentage of
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214 S. D. Perreault and A. M. Cancel
AR
AI
Fig. 4. Confocal micrograph of two rat spermatozoa stained with
flourescein isothiocyanate–peanut agglutin (FITC–PNA). FITC–PNA
stains the apical acrosomal region of rat spermatozoa with intact
acrosomes (AI). Spermatozoa that do not have an acrosome (AR)
do not show the fluorescent signal. Scale bar represents 5 µm.
spontaneously acrosome-reacted spermatozoa were found
when comparing samples from control and treated rats
(Oberländer et al., 1996). Therefore, the mode of action
appears to be through altered sperm motion and not
inhibition of acrosome reaction.
In vitro fertilization in rats
The first report of successful IVF of intact rat oocytes was
published more than 25 years ago (Miyamoto and Chang,
1973). Unfortunately, although the rat is the animal model
most extensively used for toxicology studies, limited ad-
vances have been made towards standardizing IVF in rats,
and inconsistent rates of fertilization with high inter-
experiment variability have been reported (Perreault and
Jeffay, 1993). Therefore, it is likely that IVF conditions
have not been optimized.
In vitro fertilization can be used to evaluate the effects
of chemicals on the fertilizing ability of rat spermatozoa
after exposures either in vivo or in vitro. If breeding studies
indicate that fertilization did not occur, IVF assays can be
used to identify specific deficits in sperm function and to
distinguish between such effects and infertility due to
impaired breeding or poor sperm transport.
Holloway et al. (1988) used rat IVF to evaluate the fer-
tilizing ability of spermatozoa recovered from animals ex-
posed acutely to the well-known testicular toxicant
ethylene glycol monomethyl ether (EGME). Rat oocytes,
obtained from immature female rats that had been treated
with hormones to induce ovulation, were inseminated
with rat spermatozoa and evaluated for fertilization after
an overnight incubation. Fertilization (confirmed by the
presence of a decondensing sperm head or male pronu-
cleus and sperm tail inside the oocyte) was reduced in a
dose–response manner in animals exposed to EGME
(50–200 mg kg–1). Similar effects were seen in male rats
given a single exposure to a second testicular toxicant,
1,3-dinitrobenzene, and evaluated subsequently over time
(Holloway et al., 1990). Although the highest dose affected
sperm numbers, the percentage of motile spermatozoa and
IVF, the lowest dose inhibited IVF but did not alter sperm
velocity. The same toxicants also inhibited fertilization of
zona-free oocytes when administered to rats at similar and
lower dosages to those that did not alter the percentage of
motile spermatozoa (Berger et al., 2000). Thus, IVF may be
detecting subtle effects on sperm function.
Spermatozoa must undergo spontaneous acrosome re-
action in culture and be capable of fusing with the oocyte
membrane to fertilize zona-free oocytes. In contrast, sper-
matozoa must bind to the zona, undergo zona-induced
acrosome reaction and be capable of penetrating the zona
and fusing with the oocyte membrane to fertilize zona-
intact oocytes. Thus, these two assays measure different
aspects of sperm function. Evaluation of sperm fertilizing
ability with both zona-intact and zona-free oocytes, cou-
pled with CASA analysis to determine both the percentage
of motile spermatozoa and the quality of sperm motion
(including hyperactivation during IVF), may allow differen-
tiation among antifertility effects due to alterations in ca-
pacitation, sperm motion (hyperactivation) or the ability to
undergo the acrosome reaction in vitro (Perreault, 1989).
If they are carefully designed and properly standard-
ized, IVF methods can be valuable in identifying the site of
action of impaired gamete function, and distinguishing
between effects on capacitation or zona penetration.
However, these methods are costly in terms of labour and
animal usage, and should therefore be reserved for an-
swering specific questions. However, assays for sperm hy-
peractivation and acrosome reaction are easier to conduct,
do not require the use of female rats, and can provide
valuable information about the potential effects of toxi-
cants on sperm capacitation and the acrosome reaction.
Artificial insemination
An in vivo fertility test using artificial insemination (AI) with
limited numbers of spermatozoa has proven useful in eval-
uating the impact of low dose exposure to drinking water
disinfection by-products on fertility in rats (Klinefelter et al.,
1994). This modified AI test is based on the premise that
below a threshold number of spermatozoa, successful
conception depends upon the quality of the spermatozoa
inseminated. Insemination of the threshold number of sper-
matozoa (5.0 ⫻ 106) directly into the uterus provides opti-
mal sensitivity for detecting toxicant-induced effects.
Female rats must be inseminated near the time of ovula-
tion. Oocytes can be recovered on the next day to score
fertilization, or pregnancy can be allowed to become
established so that implantation sites and fetuses can be
evaluated later and fertility, defined as the proportion of
implants, can be determined at mid- or late gestation.
An advantage of the AI test is that sperm fertilizing
ability can be evaluated in vivo under physiological condi-
tions in the female rat reproductive tract. As with IVF,
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 Measures of rat sperm function in toxicology studies
infertility or subfertility can be the result of insufficient num-
bers of healthy, viable spermatozoa inseminated (for exam-
ple, if the treatment affects sperm viability) or of specific
deficits in sperm function. Thus, this test is also sensitive to
changes in sperm motility and survival, which makes it oc-
casionally difficult to unscramble the mechanism or mode
of toxicant action. However, when sperm motility is affected
by treatment, the concentration of spermatozoa inseminated
can be adjusted upward in the samples from treated rats so
that equal numbers of motile spermatozoa can be insemi-
nated for control and treated samples.
Genetic integrity of spermatozoa
Recent interest in and concern about toxicant-induced
male-mediated developmental abnormalities makes it de-
sirable to add tests of genetic integrity to reproductive toxi-
cology studies (reviewed in Perreault, 1998). Tests for
detecting alterations in sperm chromatin structure, DNA
damage in spermatozoa (as induced by oxidative stress or
DNA alkylating agents), and chromosomal breakage or
aneuploidy in spermatozoa are being used to augment
human studies (Perreault et al., 2000) and are being
adapted for use with rat spermatozoa. A detailed discus-
sion of this subject is beyond the scope of this paper but it
has been extensively reviewed by Evenson (1999).
Conclusion
Measures of rat sperm production and function are now
being obtained routinely in multigenerational test protocols,
as well as many short-term tests and toxicology studies. Their
significance lies in the extent to which they improve the sen-
sitivity of the overall test, and provide information about the
site and mode of action and gender specificity of the test
chemical. Specific tests of rat sperm function have been ap-
plied successfully in a number of toxicology studies, and
their value with respect to identifying modes and mecha-
nisms of toxicant action should become more evident as
they are optimized and more widely used.
The information in this document has been funded by the US
Environmental Protection Agency and the EPA/UNC Toxicology
Research Program, Training Agreement CT 902908, with the
Curriculum in Toxicology, University of North Carolina, Chapel
Hill. It has been reviewed by the National Health and
Environmental Effects Research Laboratory, US Environmental
Protection Agency and approved for publication. Approval does
not signify that the contents reflect the view of the Agency, nor
does mention of trade names or commercial products constitute
endorsement or recommendation for use.
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